Method for the production and purification of bovine leukemia virus GP51 surface glycoprotein in the absence of bovine serum

ABSTRACT

The subject of the present invention is a method of obtaining purified BLV gp51 antigen as well as a novel ELISA assay using said antigen. The present invention is useful in the diagnosis of enzootic leukaemia in cattle.

This application is a §371 national stage entry of PCT InternationalApplication No. PCT/PL2009/050027, filed Sep. 27, 2009, claimingpriority of Polish Application No. PL386176, filed Sep. 29, 2008, theentire contents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The subject of the present invention is a method of obtaining purifiedBLV gp51 antigen as well as a novel ELISA assay using said antigen. Thepresent invention is useful in the diagnosis of enzootic leukemia incattle.

BACKGROUND OF THE INVENTION

Enzootic bovine leukemia (EBL) is an infectious disease of cattle causedby a type C retrovirus, BLV (Bovine Leukemia Virus) and consists oflymphatic node hypertrophy. The disease is characterized by a longincubation period (up to 7 years) and, in most cases, (ca. 60%) proceedswithout symptoms. Lymphatic cysts form in a portion of adult cattle(10-30%), whereas 1-10% develop lymphatic sarcomas on various internalorgans, with a marked increase of B lymphocytes.

A strong immune response occurs in infected individuals, which is usedin serological diagnostics. Despite the fact that the titer of anti-BLVantibodies increases as the disease progresses, they are not able tohalt the infection. Because the disease develops asymptomaticallythrough a long initial phase, the only effective method of preventingtransmission are frequent serological diagnostics and the elimination ofinfected animals. Serological assays are performed on animals over 6months, when maternal antibodies have begun disappearing. The virusitself can be detected in isolated peripheral lymphocytes using anelectron microscope, and viral DNA can be diagnosed using PCR.Immunological assays are used most frequently to diagnose bovineleukemia: gel immunodiffusion (ACID), immunoenzymatic assays (ELISA) aswell as radioimmunological detection. These methods make use ofantibodies against the antigenic proteins gp51 and p24.

BRIEF SUMMARY OF THE INVENTION

ELISA assays for diagnosing enzootic bovine leukemia are based on thedetermination of anti-BLV antibody levels in serum or milk. To constructthe assay kit it is necessary to produce purified gp51 viral surfaceantigen. This antigen is produced in a culture of FLK cells infectedwith BLV, Cell cultures make frequent use of calf serum (FCS) containingbovine immunoglobulins that, despite tedious purification procedures,contaminate the resulting preparations with bovine antibodies, whichleads to false positive results.

Available literature describes a series of labor-intensive methods ofpurifying the gp51 antigen, encompassing precipitation, extraction,centrifugation in a saccharose gradient, gel and ion exchangechromatography (Grunboeck M. et al., Polskie Archiwum Weterynaryjne1986, 24, 327-336). Ukrainian patent (UA 68930 A) describes a culturemedium containing avian serum (instead of bovine). The production of thepurified antigen, however, requires the removal of a large quantity ofballasting avian antigens.

The unsolved problem in prior art are the difficult, tedious andexpensive methods of purifying the antigen to be used in the ELISAassay. At the same time, due to the problems at this stage, contaminatedantigen preparations are produced, which, when used in an ELISA assay,lead to erroneous results and make rapid and effective diagnostics ofthe disease impossible.

DETAILED DESCRIPTION OF THE INVENTION

The subject of the present invention is a method of obtaining pure BLVgp51 antigen characterized in that the antigen production process usesculture media totally devoid of bovine serum.

In a preferential embodiment of the present invention, culture media forFLK-BLV cells are used.

In the next preferential embodiment of the present invention, theculture media encompass HyQ® SFM4MegaVir™, HyQ® PF-Vero™, and Gibco®OptiPRO™ SFM.

The next subject of the present invention is the use of purified BLVgp51 antigen in a gel immunodiffusion assay or an ELISA assay.

The next subject of the present invention is a novel ELISA assay fordetecting enzootic leukemia in cattle, characterized in that it containsa highly pure BLV gp51 BLV antigen obtained from cell culture mediatotally devoid of bovine serum.

An example embodiment of the present invention, which does not exhaustthe scope of its protection, is given below.

EXAMPLE

FLK-BLV cells are cultured in dishes or culture flasks in DMEM mediumwithout bovine serum, for example HyQ® SFM4MegaVir™, HyQ® PF-Vero™, andGibco® OptiPRO™ SFM. The culture is maintained for 2-3 days until thecells reach a large density, and then for the subsequent several days,the medium is collected from above the cells and is maintained at −20°C. The collected medium is condensed 10-fold via ultrafiltration usingan Amicon YM10 membrane and dialysed against 20 mM Tri-HCl pH 7.5,whereafter it is purified in a DEAE-Sepharose® FE column equilibratedwith 20 mM TrisHCl pH 7.5 and eluted with a sodium chloride gradient(0-500 mM). Antigen gp51 containing fractions are pooled, dialysedagainst PBS and stored at −20 C.

The antigen thus obtained can be used in gel immunodiffusion assays orELISA assays.

The invention claimed is:
 1. A process for producing purified BLV gp51antigen that is useful in an ELISA assay for diagnosing enzootic bovineleukemia comprising i) culturing FLK-BLV cells in cell culture mediumthat is totally devoid of bovine serum, wherein the culture of FLK-BLVcells is maintained for 2 to 3 days, and wherein the FLK-BLV cellsproduce BLV gp51 antigen; ii) collecting the medium from the aboveFLK-BLV cells containing the BLV gp51 antigen produced by the FLK-BLVcells; iii) concentrating the BLV gp51 antigen in the medium at least10-fold by ultra-filtration; iv) dialyzing the condensed BLV gp51antigen against 20 mM Tris-HCl pH 7.5; and v) purifying the BLV gp51antigen in a column comprising an anion exchange medium, wherein the BLVgp51 antigen is eluted from the column with a sodium chloride gradient.2. The process of claim 1, further comprising the step of dialyzing theeluted BLV gp51 antigen of step v) against PBS.
 3. The process of claim1, wherein the sodium chloride gradient is 0 to 500 mM.
 4. The processof claim 1, further comprising the step of maintaining the mediumcollected in step ii) at −20° C. prior to performing step iii).
 5. Theprocess of claim 1, wherein condensing the BLV gp51 antigen in step iii)comprises a membrane.